Complement is activated by elevated IgG3 hexameric platforms and deposits C4b onto distinct antibody domains

IgG3 is unique among the IgG subclasses due to its extended hinge, allotypic diversity and enhanced effector functions, including highly efficient pathogen neutralisation and complement activation. It is also underrepresented as an immunotherapeutic candidate, partly due to a lack of structural information. Here, we use cryoEM to solve structures of antigen-bound IgG3 alone and in complex with complement components. These structures reveal a propensity for IgG3-Fab clustering, which is possible due to the IgG3-specific flexible upper hinge region and may maximise pathogen neutralisation by forming high-density antibody arrays. IgG3 forms elevated hexameric Fc platforms that extend above the protein corona to maximise binding to receptors and the complement C1 complex, which here adopts a unique protease conformation that may precede C1 activation. Mass spectrometry reveals that C1 deposits C4b directly onto specific IgG3 residues proximal to the Fab domains. Structural analysis shows this to be caused by the height of the C1-IgG3 complex. Together, these data provide structural insights into the role of the unique IgG3 extended hinge, which will aid the development and design of upcoming immunotherapeutics based on IgG3.


Subtomogram averaging of IgG3 Fc platform
All particles used in this section are deposited in the Electron Microscopy Public Image Archive (EMPIAR) with accession code EMPIAR-11406. For the initial template, which was used during the reconstruction of the IgG3 Fab and Fc regions, a bilayer membrane model was used. This model was filtered to 40 Å and white noise was added in EMAN2 using the command e2proc3d.py. Further reconstructions were performed in Dynamo (version 1.1.157) run in MATLAB (version R2021a) 1 (Supplementary Fig. 7a). In Dynamo, particles were binned by 2 and aligned over 4 iterations to the initial model, allowing particles to fully rotate around the z and x axes with sampling discretisation of 30°, a refinement of 5 and refine factor of 2. For this first step, to align all manually picked particles to the surface, no azimuth rotation was allowed, and the shift limitations were set to 12 pixels in each direction from the centre of the particle box. Other than these parameters, the default values in Dynamo were used for global refinement. Next, the alignment of the particles was refined by also allowing the particles to azimuthally rotate over a range of 360° with sampling every 30°, a refinement of 5, refine factor of 2, and rotation around the z and x axes over a range of 20° sampling every 10°. Afterwards, multiple iterations with rotation around the z and x axes over a range of 5° with sampling every 2°, and decreasing azimuthal rotation ranges from 360° with sampling every 30° to 30° with sampling every 5°, to align particles precisely to the lipid membrane. For all these steps, the refine was set to 5 with a refinement factor of 2. Next, the 40 Å lowpass-filtered initial model was again used as a template and the previously estimated positions were used for final alignment, letting the particles rotate fully around their x and z axes with a sampling discretisation of 30° for 6 iterations. This step was calculated with refine of 5 and a refine factor of 2. Afterwards, rotations around the x and z axes were skipped, but the azimuthal rotation was set to 360° with a sampling of 30° for 6 iterations, followed by 6 iterations with azimuthal rotation of 180° with sampling every 20° and another 6 iterations with azimuthal rotation of 30° with sampling every 5°. For all iterations, refine was set to 5 with a refine factor of 2. These steps were followed by a final alignment round of 3 iterations with a rotation range around the x and z axes set to 45° and sampling every 5°, as well as an azimuth rotation range of 30° with sampling every 5°, with the refinement set to 4 and a refine factor of 2 and shift limits set to 30 pixels. The resulting average yielded the overall IgG3 map, which was dominated by the Fab-membrane density (top orange red map; Supplementary Fig. 7a).
To refine the Fc domain, we generated a smaller spherical mask centred on the Fc region with a radius of 40 pixels and a Gaussian filter of 5 in Dynamo, which was used for classification. The previously estimated particle positions were used during multi-reference alignment to limit the membrane dominating classification. The final average from the previous refinement was copied 6 times and noise with an amplitude of 5 was added in Dynamo. The classification was started by letting the particles rotate 45° around their x and z axes with sampling every 5°, azimuthal rotation of 30° with sampling every 5°, and refinement set to 5 with refine factor of 2 for 3 iterations. For the following iterations, rotations around the x and z axes were skipped and particles were allowed to azimuthal rotate within 10° with sampling every 2°, refine was set to 4 and the refine factor to 2 for 3 iterations, followed by another 3 iterations with an azimuthal rotation range of 6° with a sampling of 1°, refine of 3 with a refine factor of 2. This classification was finalised with 3 additional iterations, with an azimuthal rotation range of 2° and sampling every 0.5° and refinement of 2 with refine factor set to 2. Shift limitations were also decreased from 30 to 2 pixels in all directions over 12 iterations, yielding the classes shown in Supplementary Fig. 7a.
Multi-reference alignment resulted in a class presenting a clear Fc platform containing 571 particles (dark cyan map; Supplementary Fig. 7a). This class was used for further refinement. For the final average of the Fc region, the particles were divided into 2 half datasets, even and odd, and two independent alignments were performed using the previously calculated average as a template and the estimated particle positions and orientations. A small elliptical mask centred on the Fc region with 50 pixels in x and y, and 30 pixels in z, with a Gaussian filter of 5 was generated in Dynamo. For both datasets, 30 iterations were performed. During the first 6 iterations, the particles were allowed to rotate around their x and z axes within a range of 10° with sampling every 2° to limit the Fc platform rotating away from the membrane, which was outside the mask. The azimuth rotation range was set to 360° with sampling every 45°, with the refine set to 5 with refine factor of 2. The shift limits were set to 4 × 4 × 8 pixels in x, y and z. For the next 12 iterations, rotations around the x and z axes were skipped and only azimuthal rotations were calculated. Initially, rotations were set to 180° with sampling every 30° with a refine of 4 and refine factor set to 2 for 10 rounds. The shift limits were set to 4 pixels in all directions. Next, the rotations were set to 30° with sampling every 10° and refine set to 4 with the refine factor set to 2. Shift limitations were set to 2 pixels per direction. For the following 6 iterations, particles were free to rotate in a range of 10° with sampling every 2° and refine set to 4 with refine factor set to 2 and shift limits set to 1 pixel. The final 6 iterations used a rotation range of 2° with sampling every 0.5° and refine set to 4 with refine factor set to 2. The shift limits were again set to 1 pixel per direction. The resulting averages were further used to calculate the Fourier shell correlation (FSC) curves in EMAN2 ( Supplementary Fig. 7b), as described in the main text, which reached 19 Å (dark blue map). All the previously described subtomogram averaging steps in Dynamo were done without applying symmetry. However, there was clear C6 symmetry in the final average ( Supplementary Fig. 7c), and so the above was repeated on the 571 even/odd half-datasets but with C6 symmetry applied. For this C6symmetrised map, the FSC reached 14 Å (magenta map; Supplementary Fig. 7).
Classification and subtomogram averaging of IgG3-C1-C4b All particles used in this section are deposited in EMPIAR with accession code EMPIAR-11407. The initial model of the IgG3-C1-C4b map described in the main Methods section was used for subtomogram averaging in Dynamo ( Supplementary Fig. 10b), where, if not mentioned differently, default values were used. Particles were binned by 2 and aligned to this model over 4 iterations, allowing particles to fully rotate around the z and x axes with sampling discretisation of 30°. No azimuthal rotation was allowed. Refine was set to 5 with a refine factor of 2. Shift parameters were set to 12 pixels in each direction from the centre of the particle box, and a spherical mask with a radius of 110 pixels, generated in Dynamo, was used. The resulting average was used as a template and the estimated particle coordinates as a table for the following alignment. This alignment was split into 5 rounds and a spherical mask with a radius of 78 pixels was used. The first round contained 3 iterations and particles were binned by 4. The rotation range around the particles' x and z axes as well as the azimuth rotation range were set to 360° with sampling every 60° and refine was set to 5 with refine factor of 2. Shift limits for this round were set to 12 pixels in each direction. The second round contained another 3 iterations and particles were binned by 2. Now, particles were allowed to rotate within a range of 20° with sampling every 5° around their x and z axes. The azimuth rotation range was set to 60° with sampling every 5° and the refine was set to 2 with a refine factor of 2. The shift limits for this round were set to 4 pixels in each direction. The next round contained 3 iterations and the particles were again binned by 2. This time, the rotation around the x and z particle axes and the azimuth rotation range was set to 4° with a sampling of 2° and the refine set to 2 with refine factor set to 2. The shift limits were set to 2 pixels per direction using particles binned by 2. The fourth round contained 5 iterations with rotation ranges for their x and z axes and the azimuthal rotation range set to 4° with sampling every 2°, and the refine set to 3 with refine factor set to 2. For these iterations, the particles were binned by 2, the shift limits were set to 2 pixels in each direction. For the last round in this refinement, unbinned particles were used. The rotation range for rotating the particle around their x and z axes as well as the azimuth rotation range was set to 4° with sampling every 2° and refine set to 3 with the refine factor set to 3. The shift limits were set to 2 pixels in each direction.
Next, multireference classification was performed over 4 rounds by cloning the resulting average 3 times and adding noise with an amplitude of 3 in Dynamo. A spherical mask with a radius of 110 pixels was used for this classification. During the first 3 iterations, particles were binned by 4 and allowed to rotate within a range of 20° around their x, z and azimuthal axes, with a sampling of 5°. Refine was set to 5 with the refine factor of 2 and shift limits were set to 4 pixels in each direction. For the next 3 iterations, particles were binned by 4 and the rotation around the x and z axes of the particles and the azimuth rotation range were set to 4° with sampling every 2°. Refine was set to 2 with a refine factor of 2, and the shift limits during this round were set to 2 pixels per direction. For the next 5 iterations, particles were binned by 2 and allowed to rotate around their x and z axes and to azimuthally rotate within a range of 4° with sampling every 2°, with a refine set to 3 with a refine factor of 2. The shift limitations were set to 2 pixels in each direction. For the final 5 iterations in this multireference refinement, the particles were used unbinned. The rotation range around the particles' x and z axes as well as the azimuth rotation range were set to 4° with sampling every 2°. Refine was set to 3 with a refine factor set to 1.
Shift limits were set to 1 pixel per direction. This classification resulted in 2 classes with clear C1 complexes bound to antibodies on a membrane, which contained 1,612 and 816 particles (pastel lime and pastel blue maps, respectively; Supplementary Fig. 10b).
The two classes resulting from multireference refinement were combined, and the remaining 2,428 particles were used for all following refinements ( Supplementary Fig. 10b). The dataset was split into 2 half datasets, even and odd, before the 2 independent alignments were performed. The initial model from the beginning was used, together with a spherical mask of radius 110 pixels. For the even and odd refinements, 5 rounds, each containing 6 iterations were performed. For the first round, particles binned by 2 were used and were allowed to rotate azimuthally within a range of 180°, sampling every 30°. The refine was set to 5 with a refine factor of 2. Shift limits were set to 4 pixels per direction. For the second round, particles were again binned by 2 and the azimuth rotation range was set to 30° with sampling every 10° and refine set to 4 with a refine factor of 2, and shift limits were set to 2 pixels per direction. During the third round, particles binned by 2 were allowed to rotate azimuthally within a range of 10° and sampling every 2°. The refine was set to 3 and the refine factor set to 2, and the shift was limited to 1 pixel per direction. For the fourth round, unbinned particles were used that were allowed to rotate in an azimuth rotation range of 2° with sampling every 0.5°. Refine was set to 2 with a refine factor of 2, and the shift limits for this as well as for the next round were set to 1 pixel per direction. The fifth round was performed on unbinned particles. The azimuth rotation range was set to 1° with sampling every 0.5° and refine set to 1 with refine factor of 2. All refinements were performed without applying symmetry. This resulted in an overall IgG3-C1-C4b map, which reach 34 Å resolution (sky blue map; model 2, Supplementary Fig. 10b).
Next, we performed multiple focused refinements to attempt to improve the resolution of different parts of the structure. We used the overall IgG3-C1-C4b map from above as an initial model and the estimated particle positions and orientations. First, we focussed on the C1 complex. We generated a spherical mask with a radius of 65 pixels centred on the C1 complex ( Supplementary Fig. 10b). We used all 2,428 particles split into even and odd datasets. These 2 independent refinements were performed over 14 iterations. For the first 2 iterations, particles were binned by 2 and these were allowed to rotate within a range of 30° around their x and z axes with sampling every 5°. The azimuth rotation range was set to 360° with sampling every 45° and refine was set to 5 with refine factor set to 2. The shift limits were set to 4 pixels per direction. For the next 2 iterations, particles were again binned by 2. These particles were then allowed to rotate around their x and z axes within a range of 10 and sampling of 2°. Additionally, these particles were allowed to rotate azimuthally within a range of 180° and sampling of 30°. Refine was set to 5 with a refine factor of 2 and shift limits were set to 4 pixels per direction. The following 4 iterations used particles binned by 2 with a rotation range around the x and z axes and azimuth rotation range both set to 10° with sampling every 2°, and the refine was also set to 2 with refine factor of 2. The shift limit was set to 2 pixels in each direction. For the next 4 iterations, unbinned particles were used, which were allowed to rotate around their x and z axes over a range of 2° with sampling every 0.5°. The azimuth rotation range was set to 2° with sampling every 0.5°. Refine was set to 1 with the refine factor set to 2 and a shift limit of 1 pixel per direction was allowed. For the final 2 iterations, unbinned particles were allowed to rotate around their x and z axes and azimuthally rotate within a range of 1° with sampling every 0.5°. The refine was set to 1 with a refine factor of 2 and the shift limits were set to 1 pixel per direction. The resulting IgG3-C1-C4b focussed on the C1 region map can be seen in Supplementary Fig. 10b,c (dark green map; model 3).
To yield insights into how the proteases are oriented within the C1 complex, we generated a mask with a diameter of 46 pixels and a height of 26 pixels centred on the protease domain which included the C1 globular head regions (Supplementary Fig. 10b). We used the previously generated IgG3-C1-C4b focussed on the C1 region map as a template. The particles were again split into even/odd datasets, each dataset containing the same particles as the previous halfdatasets. Particles were binned by 2 and over 6 iterations allowed azimuth rotation over a range of 30° with a sampling of 5°, refine was set to 5 with the refine factor set to 2. The shift limits were set to 4 pixels per direction. For the next 6 iterations, particles binned by 2 were free to azimuthally rotate within a range of 10° with a sampling of 2°. Refine was set to 2 with the refine factor set to 2 and the shift limits were set to 2 pixels per direction. For the final 6 iterations, unbinned particles were allowed to azimuthally rotate within a range of 2° with a sampling of 0.5°. Refine was set to 1, the refine factor was set to 2 and the shift limits were set to 1 pixel per direction. This average resulted in the focussed C1 globular head region (Supplementary Fig. 10b; purple map; model 4).
Next, we focussed on the antibody Fc platform. For this focused refinement, we generated a mask with a radius of 50 pixels centred on the Fc region ( Supplementary Fig. 10b). Model 2 was used as an initial model. The same rotation scheme as for the focussed C1 refinement described above was used, but by using the Fc-specific mask. This resulted in the IgG3-C1-C4b focussed on the Fc region map (Supplementary Fig. 10b; dark red map; model 5).
The resulting average and refined particles were used to focus the refinement further to the Fab-C4b region. Therefore, we generated a cylindrical mask with a radius of 35 pixels and height of 80 pixels with a Gaussian filter of 5 centred on the C4b-Fab region ( Supplementary Fig. 10b). The same even and odd datasets from Fc focussed model above were used. For the first 5 iterations, we used binned by 2 particles, that were free to rotate around their x and z axes and azimuthally rotate within a range of 30° with a sampling of 10°. Refine was set to 5 with a refine factor of 2 and the shift limits were set to 4 pixels in each direction. The following 5 iterations again used particles binned by 2, which could rotate around their x and z axes within a range of 10° with a sampling of 2°. The azimuth rotation range was set to 10° with sampling every 2°. Refine was set to 5 with a refine factor of 2 and shift limits of 2 pixels per direction. The next 5 iterations also used particles binned by 2. These particles were allowed to rotate around their x and z axes and azimuthally rotate within a range of 2° with a sampling of 0.5°. Refine was set to 2, with a refine factor of 2 and a shift limitation of 2 pixels per direction. For the final 2 iterations, unbinned particles were used. Particles were allowed to rotate around their x and z axes as well as azimuthally rotate within a range of 1° with sampling every 0.5°. Refine was set to 1 with a refine factor of 2 and shift limits were set to 1 pixel per direction. These rounds of refinement resulted in the IgG3-C1-C4b focussed on the C4b region map (Supplementary Fig.  10b; ochre yellow map; model 6). The focused refinements around the C1-Fc region, C1rs platform, and Fab-C4b region including the used masks are shown in Supplementary Fig. 10b. FSC for each refinement were calculated using EMAN2 after aligning the two even/odd halfmaps and applying a tight mask generated in e2filtertool.py from the EMAN2 suite ( Supplementary Fig. 10c).

Modelling the C1 complex
The six C1q globular head domains (gC1q) were fit into model 3 ( Supplementary Fig. 10b) as rigid bodies using 1PK6 2 as a model. The heterotrimeric gC1q domain is approximately spherical, and so known orientation from PDB model 6FCZ 3 was used to orient the domains in the map ( Supplementary Fig. 17), such that chains B and C were adjacent to the IgG3-Fc region, as previously shown 3,4 . The collagen arms of C1q are composed of six heterotrimeric collagen fibrils, which were modelled using ccbuilder 2.0 5 using default values for radius and pitch. The region of the collagen arms between the C1r2s2 protease platform and gC1q domains, comprising residues A58-89, B60-91 and C57-88 (A, B and C refer to the individual chain within the heterotrimeric structure), were fit into the map as rigid fibrils between the relevant gC1q domain and their locations in the protease platform, which bind via known lysine residues 6 . Next, the C1q stalk, comprising residues A1-39, B1-41, and C1-38 were fit into the map and known disulfide bonds formed between the C-C and A-B interchain cysteine residues. The C1q stalk and collagen region between the C1r2s2 platform and gC1q domains are separated by residues A40-57, B42-59 and C39-56, which were oriented in the map to connect these two regions. The complete C1q structure was formed by forming bonds between the termini using ISOLDE within UCSF ChimeraX 7, 8 , which were then allowed to relax in a brief simulation within the map to yield the final model of C1q in Fig. 3e. The C1r2s2 proteases were modelled based on the crystal structure of the CUB1-EGF-CUB2 ( Supplementary Fig. 1) heterotetramer with PDB code 6F1C 9 . The C1r protease arms, comprising the CCP1-CCP2-SP domains, were based on PDB model 1GPZ 10 . These were fit into the map as rigid bodies and linked to the relevant CUB2 domains using ISOLDE. The C1s protease arms were modelled based on PDB model 4J1Y 11 . The different orientations of C1s were modelled by rotating the CUB2 domain around the C1q collagen arms as described in the main text. These were also linked together using ISOLDE to form the complete C1r2s2 heterotetramer. The C1q and C1r2s2 models were placed in the map together and briefly simulated in ISOLDE to yield the complete C1 complex.

Supplementary Figures
Supplementary Fig. 1. Overview IgG1, IgG3 and classical complement pathway. MS/MS analysis was performed on the quadruply charged species at m/z 801.666 [M+4H] 4+ . C-terminal fragments (y-ions) are annotated for both the tryptic peptide from the Fc region of (a) IgG3 (green) or (b) IgG1 (red) and the C4b peptide (orange, Q from thioester). Crosslinks between the glutamine of C4b and the lysine at position 328 of IgG1 and lysine at position 375 IgG3 were observed, based on the fragmentation pattern. Characteristic fragments for the crosslink to K328 and K375 for IgG1 and IgG3, respectively, are represented by y4-y8 ions of the VSNKALPAPIEK peptide. The crosslinked peptide eluted at 32.22 min and 32.12 min for IgG1 and IgG3, respectively (Fig. 4d). All cysteines are carbamidomethylated and the methionine in the C4b peptide is oxidised, indicated as m.

Supplementary Tables
Supplementary Table 1. BLAST comparison of IgG1 and IgG3 constant heavy chain sequences. The upper hinge, disulphide-linked core and lower hinge are highlighted as yellow, cyan and green, respectively. Sites that are linked to C4b are shown in bold and are underlined. IgG3m5 specific amino acids are indicated in red and the highly conserved N297 residue with the N-linked glycan is indicated in magenta. Residues follow Eu numbering. Gaps and "+" indicates non-conservative and conservative mutations, respectively.